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Downloaded from http://www.jci.org on June 18, 2017. https://doi.org/10.1172/JCI109772 Circadian Rhythm of Intestinal Sucrase Activity in Rats MECHANISM OF ENZYME CHANGE MARK A. KAUFMAN, HELEN A. KORSMo, and WARD A. OLSEN, Gastroenterology Research Laboratory, William S. Middleton Memorial Veterans Administration Hospital and Department of Medicine, Center for Health Sciences, University of Wisconsin, Madison, Wisconsin 53706 A B S T R A C T Past investigation has revealed that the circadian rhythm of intestinal sucrase activity in rats is primarily cued by the time of feeding. We examined the mechanism of the circadian rhythm by methods involving quantitative immunoprecipitation of sucraseisomaltase protein and study of decay of radioactively labeled protein. Rats were placed on a controlled feeding regimen (1000- 1500 h) and then sacrificed at 3-h intervals over a 24-h period. Immunotitration experiments indicated that the circadian rhythm was the result of changes in the absolute amount of sucrase-isomaltase protein present and not of changes in the enzyme's catalytic efficiency. To study the mechanism of this circadian variation in sucrase-isomaltase mass, [14C]sodium carbonate was injected and, after maximum incorporation into brush border protein, the rats were sacrified at 3-h intervals. Sucrase-isomaltase protein was isolated by immunoprecipitation, and the decrease in total disintegrations per minute over time was used to study degradation of the protein. Enzyme degradation was not constant but exhibited a clear circadian rhythm. The period of increasing enzyme mass was characterized by virtual cessation of enzyme degradation (t1/2 of 38 h), and the period of declining enzyme mass by rapid degradation (t1/2 of 6 h or less). We found similar changes in enzyme degradation in fasted animals, demonstrating that the changes were not the result of decreased isotope reutilization during feeding. We found no evidence of a circadian rhythm in [14C]leucine incorporation into the protein, suggesting that enzyme synthesis was constant. These results indicate that the circadian rhythm of sucrase activity represents changes in the total amount of enzyme protein that are, at least in large part, secondary to changes in the enzyme's degradation rate. INTRODUCTION A number of studies have demonstrated a circadian rhythm in the activities of intestinal disaccharidases (1-5). That the circadian rhythm of sucrase activity in rats is synchronized by the time of feeding, but not by the lighting rhythm, has also been clearly established (2-5). A significant rise in enzyme activity usually occurs 1 h before onset of feeding with continued elevated levels throughout the feeding period. Activity begins to decrease within 1-3 h after feeding ceases. This rhythm can be temporally shifted by altering the time of controlled feeding (5), but can be eliminated only by several days of starvation (2). Because most investigators have found a rise in enzyme activity 1 h before the scheduled beginning of feeding, many have postulated that it is the anticipation of feeding that triggers the rise in enzyme activity rather than food intake itself. This suggestion is supported by the observation that the rhythm persists for at least 2 d during starvation (2). We studied the mechanism of this circadian rhythm of sucrase activity. We used quantitative immunoprecipitation to determine whether increased enzyme activity during feeding resulted from an absolute increase in the amount of enzyme protein present or from a change in the proportions of active and inactive enzyme. We also studied incorporation of isotopically labeled precursor into sucrase-isomaltase protein to look for evidence of a circadian rhythm of either enzyme degradation or synthesis. Our findings indicate that the circadian rhythm of intestinal sucrase activity represents changing enzyme protein levels that appear to be the consequence of changing degradation rates. METHODS Received for publication 24 January 1979 and in revised Male Sprague-Dawley rats (Madison, Wis.) weighing 75150 g were placed on a controlled feeding regimen made form 18 January 1980. 1174 J. Clin. Invest. (© The American Society for Clinical Investigation, Inc. * 0021-9738/80105/1174/08 $1.00 Volume 65 May 1980 1174-1181 Downloaded from http://www.jci.org on June 18, 2017. https://doi.org/10.1172/JCI109772 available from 1000 to 1500 h only, with a standard diet that included carbohydrate. After 1-2 wk on this feeding regimen, animals were sacrificed at appropriate intervals for study of solubilized sucrase-isomaltase as described below. Enzyme solubilization. The following procedures were done at 4°C. The animals were stunned, decapitated, and the abdominal cavity opened with prompt removal of the small intestine from the ligament of Treitz to the cecum. The intestine was rinsed with normal saline, flushed with saline, everted, rinsed again, and then scraped over ice with a glass slide to remove the mucosa. After adding 0.1 M potassium phosphate buffer pH 7.4 (5 ml/g mucosa), mucosa from each rat was hom6genized over ice for 10 and then 15 s with a Polytron homogenizer at setting No. 4 (Brinkmann Instruments, Inc., Westbury, N. Y.). Papain and cysteine were each added in amounts of 0.2 mg/ml of homogenate, and the homogenates were then incubated at 37°C for 60 min, followed by centrifugation for 60 min at 105,000 g at 4°C. (Addition of the protease inhibitor phenylmethyl sulfonyl fluoride at a concentration of 0.5 mg/ml after 60 min of incubation with papain and its inclusion during immunoprecipitation did not affect the pattern found on sodium dodecyl sulfate-acrylamide gel electrophoresis of immunoprecipitates, so that a protease inhibitor was not used.) Resultant supernatant fractions were dialyzed overnight at 4°C against 0.1 M KPO4 buffer, pH 7.4. The dialyzed supernatant fractions will be referred to as papain supernates. Recovery of homogenate sucrase activity in papain supernates was not different between periods of high and low activity (89 vs. 87%). Sucrase-isomaltase immunoprecipitation. Purification of sucrase-isomaltase for raising antibodies was done as described by Kolinska and Kraml (6). We have previously shown (7) that the purified protein was homogeneous by acrylamide gel electrophoresis at two pHs; the protein was also homogeneous by analytical ultracentrifugation. Preparation of the rabbit antiserum was done as described by Olsen and Korsmo (7). Although the antiserum precipitated 100% of sucrase activity in papain supernates under conditions of antibody excess, it precipitated no lactase or leucylnaphthylamidase activity. To isolate sucrase-isomaltase protein, the optimal relative amounts of antiserum and enzyme activity were determined by double diffusion in agar gel as described by Ouchterlony (8). Quantitative precipitin reactions were carried out as per Kabat and Meyer (9). In practice, 0.01 ml of antiserum was added to aliquots of papain supernate from two different groups of rats that had significantly different sucrase specific activities. These aliquots contained identical amounts of enzyme activity ranging from 0.05 to 0.30 U (1 U = 1 gmol of sucrose hydrolyzed/min) and were then made up to identical volumes with isotonic saline. The tubes were held at 40C for 2 d and then centrifuged at 1,000 g for 15 min at 4C. The pellet was washed three times with normal saline and then assayed for protein (10). The supermates obtained after the initial centrifugation (before washing) were assayed for sucrase activity. The quantity of protein present in the antigen-antibody precipitate and the amount of sucrase activity present in the papain supernate for each rat were plotted against the amount of original enzyme. All the above was done in duplicate. Isotopic decay of labeled sucrase-isomaltase. Proteins were labeled by tail-vein injection of 500 ACi of [14C]sodium carbonate (2-10 mCi/mmol sp act, New England Nuclear, Boston, Mass.). Unless specifically stated, the usual 10001500 h access to food was provided during these experiments. Preliminary studies indicated that maximum labeling of sucrase-isomaltase under these conditions was achieved by 14 h so that, beginning 14 h after injection, the rats were sacrificed at 3-h intervals (four rats per interval) and papain supermates prepared from individual animals. Sucrase-isomaltase protein was isolated in duplicate by adding 0.08 ml of antiserum to solutions containing 1.664 U of sucrase activ ity made up to identical volumes with saline for each rat, and the pellet obtained after washing was dissolved in 0.2 ml of Bio-Solv, formula BBS-3 (Beckman Instruments, Inc., Fullerton, Calif.). Scintillation fluid consisted of 6 ml of a solution of toluene, Liquifluor (New England Nuclear.), and alcohol (1,000:42: 379 ml, respectively). Each sample was counted for 20 min at -70% efficiency in a Packard Tri-Carb liquid scinitillation spectrometer, model 3330 (Packard Instruments Co., Inc., Downers Grove, Ill.). Acrylamide gel electrophoresis of the immunoprecipitates in the presence of sodium dodecyl sulfate with counting of gel slices demonstrated that -90% of precipitated counts migrated with sucrase-isomaltase subunits (determined by simultaneous electrophoresis of the isolated protein). Analysis of the original supernate for sucrase failed to reveal significant residual enzyme activity, indicating essentially total precipitation of the enzyme. To correct for nonspecific precipitation (11), 1.664 U of nonradioactive sucrase was added to the papain supernates after removal of imimunoprecipitates. Immunoprecipitation with 0.08 ml of antiserum was repeated, and the radioactivitv measured in the pellets. Nonspecific precipitate radioactivitY was relatively constant and averaged 44±3 (SEM) dpm, whereas the radioactivity of sucrase-isomaltase decreased markedly with time from injection. In addition, the supernate from the nonspecific immunoprecipitation was also checked for sucrase activity to ensure complete immunoprecipitation. After subtracting nonspecific precipitation, the radioactivity "disintegrations per minute" of sucrase-isomaltase was plotted against time on semilogarithmic paper. Half-lives were calculated by linear regression. Differences in slopes of lines were statistically compared with methods outlined by Goldstein (12). The radioactivity of total proteins in papain supermates was determined by the technique of Siekevitz (13). In duplicate runs, trichloroacetic acid was added to precipitate protein; nucleic acid was then removed with hot trichloroacetic acid and lipid with alcohol-ether-chloroform and ether. The final air-dried residue was dissolved in 300 ,ul of 0.3 N sodium hvdroxide for determination of protein and radioactivity. Incorporation of ["4C]leucine into sucrose-isomaltase. To provide evidence that might support a circadian rhythm in enzyme synthesis, we studied incorporation of [m4C]leucine into sucrase-isomaltase. Animals were injected with 20 iLCi of [14C]leucine (L-[1-_4C]leucine, >50 ACi/mmol sp act, New England Nuclear) by tail vein at a time of low enzyme activity (0130 h) and at a time of rising activity (0930 h). 10 and 60 min later, animals were sacrificed and sucrase-isomaltase and total papain supermate radioactivity determined as described above. Enzyme assays and statistical evaluation. Disaccharidase activity was determined by the method of Dahlqvist (14), and leucylnaphthylamidase activity as described by Porteous and Clark (15). All assays were done in duplicate. Statistical evaluation of differences between means was done by t test. Determination of specific activity offree amino acids. Aliquots of individual mucosal homogenates containing 5 mg of protein were pooled for each time point after injection of ['4C]sodium carbonate. To each milliliter of pooled homogenate, 100 ,ul of 50 g/100 ml sulfosalicylic acid was added over ice. After mixing and removal of the precipitate, the supernatant pH was adjusted to 2.2 with 4 g/100 ml lithium hydroxide and amino acid analysis performed by the method of Stein and Moore (16). Separate runs were performed for separation of amino acids for isotope counting by the methods described above. Circadian Rhythm of Sucrase Activity in Rats 1175 Downloaded from http://www.jci.org on June 18, 2017. https://doi.org/10.1172/JCI109772 RESULTS Sucrase activity. Under the controlled feeding regimen, a circadian rhythm of sucrase activity in both mucosal homogenates and papain supernates was found corresponding to the time of feeding (Fig. 1). Table I demnonstrates that enzyme activity during a 6-h period preceding feeding (0300-0900 h) is statistically different from that during a 6-h period encompassing feeding (1200-1800 h). Both total activity and specific activity were significantly elevated during feeding with P < 0.001 in each case. Iinmunoprecipitation experiments. We studied individual papain supernates from three animals sacrificed at 0600 h (low enzyme specific activity) and from three animals sacrified at 1500 h (high enzyme specific activity), (0.218+0.023 U/mg, SEM vs. 0.306+0.017 U/ mg, P < 0.02). Ouchterlony double-diffusion plate analysis (8) was performed with 0.25 ul1 of antiserum in the center well and one of the six papain supernates, each containing 0.0052 U of sucrase activity in each of the peripheral wells. We found a single precipitin band, suggesting the presence of a single antigenic protein in the six papain supernates reactive with the TABLE I Sucrase Activity of Papain Supernates Group Total activity Specific activity MmolImin/aninal pmollmnin/ng protein 8.67+0.89 0.208±0.012 16.03±+1.40 0.328±0.018 P <0.001 P < 0.001 0300-0900 h 1200-1800 h Significance of difference* Means+SEM are given. t test. * pernates. All sucrase activity was precipitated until the equivalence point was reached, (0.15 U), at which time sucrase activity in the supernate increased secondary to antigen excess. The equivalence points were the same in both groups of animals, indicating that enzyme activity per unit of immunologically identified protein was not significantly different. Isotopic decay of labeled sucrase-isomaltase. Fig. 3 demonstrates the decline in radioactivity of sucraseisomaltase in studies between 0500 and 1700 h. Total radioactivity of sucrase-isomaltase for each animal antiserum. Fig. 2 represents the results of quantitative immuno- rather than specific radioactivity is presented because precipitation reactions done with these six papain su- of the presence of nonsteady-state conditions (17). Anl abrupt change in the slope of the sucrase-isomaltase decay curve occurred at 1100 h. Before that time (during the period of rising enzyme activity), enzyme protein <--feed degradation was slow with a t112 of 38 h; thereafter 0.40(during the period of falling enzyme activity), degradation was rapid with a tl2 of 6 h. In contrast to sucraseisomaltase, degradation of total proteins solubilized by .CL papain was characterized by a single and relatively slow rate (t412 of 23 h). oi 0.30 To confirm the observation of two distinct rates of degE radation of sucrase-isomaltase and to determine the time at which the rapid degradation associated with feeding ceased, we studied the decline in labeled enzyme between 2000 and 1100 h. Fig. 4 demonstrates <)0.1 3 t 0.20 the presence of two slopes in the curve describing again < degradation of sucrase-isomaltase, with an abrupt change at about 0200 h. Thus, rapid degradation of the enzyme began at about 1100 h in association with feedC,) ing and continued until -0200 h; after 0200 h degrada0 0.10tion virtually stopped. To evaluate the direct effects of food intake on sucrase-isomaltase degradation, a similar study was per0.0 formed on fasted rats allowed free access to drinking water only. (The animals sacrificed for the initial time point had been fasting for 29 h). We found, as others 0300 0600 0900 1200 1500 1800 2100 2400 have reported (2), that a circadian rhythm of sucrase acTime (h) tivity persists even during a short-term fast. We also FIGURE 1 The circadian rhythm of sucrase specific activity in papain supemates. Enzyme activity rose during the found, as shown by Fig. 5, clear evidence that the feeding period, 1000-1500 h. Mean values from four animals rhythm continues to be accompanied by a changing ±SEM are shown. pattern of degradation. Fasting appeared to have some 1176 M. A. Kaufman, H. A. Korsmo, and W. A. Olsen Downloaded from http://www.jci.org on June 18, 2017. https://doi.org/10.1172/JCI109772 0 0. .U 0 0 V 0 L- C L. C 0. 4- .0 CL c In Ul) 0 .1; 0 L- U) Sucrose Units Added FIGURE 2 Quantitative immunoprecipitation reactions of sucrase-isomaltase in papain supernates from two groups of rats corresponding to high sucrase activity (1500 h) and low sucrase activity (0600 h). To 10 ,ul of antiserum were added papain supernate aliquots from each of six rats containing the enzyme activities indicated. Precipitation was carried out as described in Methods. The closed symbols represent the protein content of antigen-antibody precipitates and the open symbols, the enzyme activity in the supernate after removal of the precipitate. I 10 0 9 x 8 7 - tI/2 23h Total Protein ._ 065 4 E CL 3 feed > 22 W 0 x - 10 9 08 E 7 Sucrose- Isomaltase <-tfi/2 6h 6 E0. 4 effects on degradation, however, with the rapid phase somewhat slower than found during feeding and the time of virtual cessation of degradation occurring later (0500 instead of 0200 h). Fig. 6 shows the specific activities offree amino acids in mucosal homogenates during the study of sucraseisomaltase degradation shown in Fig. 3. The first time point was 14 h after injection of [14C]sodium carbonate. Specific activities of all amino acids were low and their decline with time did not seem appreciably altered during the feeding period. Incorporation of [14C]leucine into sucrase-isomaltase. Incorporation of [14C]leucine into sucrase-isomaltase was expressed relative to incorporation into total protein to correct for possible circadian changes in precursor pool size. As shown in Table II, we found no difference in leucine incorporation into sucrase-isomaltase between a period of low and rising enzyme activity, suggesting the absence of a circadian rhythm of enzyme synthesis. 10 3 DISCUSSION Theoretically, the circadian changes in sucrase activTime of Day (h) ity could be the consequence of changes in the amount FIGURE 3 Degradation of sucrase-isomaltase and papain- of enzyme protein present or changes in the proporsolubilized total proteins between 0500 and 1700 h. Mean tions of active and inactive enzyme (18). Our immunotivalues for four animals ±SEM are given. Regression lines were tration experiment, where enzyme activity was titrated calculated by the least squares method using individual values. The difference between the two slopes for sucrase- against a constant amount of antiserum (Fig. 2), demonisomaltase degradation was statistically significant (P < 0.001). strated that the equivalence points (beyond which en0200 0500 0800 1100 1400 1700 Circadian Rhythm of Sucrase Activity in Rats 1177 Downloaded from http://www.jci.org on June 18, 2017. https://doi.org/10.1172/JCI109772 9080 ~70- 60 _ 504030 o 20 E-feed- E C io1 EN9 -6 54- 3- hazards that must be discussed, The major assumptions (and potential weaknesses) in the method are that (a) the radioactivity isolated by immune precipitation represents sucrase-isomaltase rather than a contaminant and (b) there is not significant reutilization of isotope (19). Both assumptions seem valid. Immune precipitation depends upon the monospecificity of the antiserum. Our antigen was homogeneous by acrylamide gel electrophoresis in two pH systems and by analytical ultracentrifugation. The double diffusion and quantitative immunoprecipitation experiments indicate that the resultant antiserum precipitated a homogeneous protein, a conclusion supported by the observation that it did not precipitate other brush border proteins solubilized by papain such as lactase and leucylnaphthylamidase. Finally, essentially all radioactivity of the immune precipitate (after subtraction of nonspecific isotope precipitation) migrated with sucrase-isomaltase subunits on sodium dodecyl sulfate acrylamide gel electrophoresis. Thus, immunoprecipitated radioactivity represents sucrase-isomaltase. Reutilization of labeled amino acid precursor with apparent half-lives longer than actual half-lives has been a major problem with the use of isotope decay curves to study protein degradation (20). We therefore chose 2100 90 80 70 60 50 2000 2300 0200 0500 0800 1100 1400 Time of Day (h) Degradation of suierase-isomaltase between 2000 FIGURE 4 and 1100 h. Mean values for four animals ±SEM are given. Regression lines were calculated by the least squares method. The difference between the slope of'the slow component shown here (0200-1100 h) and that shown in Fig. 3 (05001100 h) was not statistically significant. The slope of the faster component in this study (2000-0200 h) was, however, different from that component in Fig. 3 (P < 0.001), with a t1/2 of 2.4 vs. 6 h. 40 CY 6 30 x D 20 E C a 10 zyme activity was found in the supernates after centrifugation) for enzyme from animals with high sucrase activity (1500 h) and low activity (0600 h) were not significantly different. This finding indicates that the circadian changes in enzyme activity were accompanied by o6 comparable changes in immunologically reactive en- * 8 7 6 5 4 3 zyme protein. The circadian rhythm of enzyme protein levels must reflect a rhythm involving enzyme synthesis, degraI 2000 2300 0200 0500 0800 1100 1400 1700 dation, or both. We studied the degradation of sucraseTime of Day (h) isomaltase protein by measuring the radioactivity of immunoprecipitated protein after pulse-labeling by in- FIGuRE 5 Degradation of sucrase-isomaltase in fasted anijection of "4C-precursor. This technique, like all tech- mals. Mean values for four animals ±SEM are given. Regresniques for assessing protein degradation, has theoretical sion lines were calculated by the least squares method. 1178 M. A. Kaufman, H. A. Korsmo, and W. A. Olsen Downloaded from http://www.jci.org on June 18, 2017. https://doi.org/10.1172/JCI109772 TIME OF DAY (hour) I I 14 17 HOURS AFTER INJECTION OF 20 23 26 [14C] SODIUM CARBONATE FIGURE 6 Specific activity of free amino acids in mucosal homogenates during study of sucrase-isomaltase degradation. A pool of homogenates from four animals sacrificed at each time point was assayed as described in Methods. Both the time of day and the time from injection of ['4C]sodium carbonate are shown. Radioactivity of amino acids not shown was negligible. 14CO3 as a precursor to minimize reutilization. This technique has been used to study protein degradation in liver, muscle, and brain (21-24), and has been suggested as a general tool for the study of turnover of tissue proteins (20). Labeling of glutamate and aspartate after "4CO3 injection occurs by carboxylation of pyruvate and the interconversions of the tricarboxylic acid cycle. As originally described for study of muscle protein metabolism (22), the radioactivity of protein asparTABLE II Relative Incorporation of Leucine into Sucrase-Isomnaltase Incorporation time Injection at 0130 h Injection at 0930 h Significance of difference* 10 min 60 min 0.017±0.003 (3) 0.025+0.004 (3) 0.043+0.004 (3) 0.042+0.007 (3) NS NS [14C]Leucine was injected (20 uCi i.v.) at 0130 and 0930 h, and the animals sacrificed 10 and 60 min later. Disintegrations per minute of sucrase-isomaltase were divided by disintegrations per minute of papain supernate total protein to obtain relative incorporation. Means+SEM are given. Numbers of animals are given in parentheses. * t test. tate and glutamate were followed. Because the carboxyl groups of aspartate and glutamate are rapidly exchanging with the bicarbonate pool (which rapidly becomes unlabeled after a single injection of 14CO3, t1,2 of 12 min), labeled carboxyl groups should be largely replaced by unlabeled groups before reincorporation. Investigators studying liver proteins, on the other hand, have only had to measure radioactivity of the entire protein because all labeled amino acids in liver exchange rapidly with the bicarbonate pool. Our study of a single intestinal protein involved measurement of the radioactivity in the entire protein rather than the aspartate and glutamate constituents. This was necessary because of the small quantities of sucrase-isomaltase protein. Swick and Handa (25) demonstrated that after feeding 14CO3, - 10% of the alpha carboxyl carbon of glutamate and aspartate in intestinal protein was derived from the CO2 pool as well as 10% of the beta carboxyl carbon of aspartate. In addition about half of the guanidino carbon of arginine and lesser fractions of the carboxyl groups of proline, serine, glycine, and alanine were derived from CO2 fixation (25). Because sucrase-isomaltase is unusually rich in aspartate and glutamate, with 110 and 90 residues per molecule, respectively (26), it seems likely that much of our measured radioactivity was, in fact, from aspartate and glutamate residues. In addition, intestinal mucosa is one of the few tissues besides liver that contains arginase in appreciable quantity (27), which should diminish reincorporation of the labeled guanidino carbon of arginine. These considerations suggest that reincorporation of isotope may not be extensive. The effect of reutilization on apparent halflife, moreover, should be minimal. As Poole (28) has demonstrated, apparent half-lives of proteins that turn over rapidly are much less affected by isotope reincorporation than for proteins that turn over more slowly. The effect of significant reincorporation of isotope on half-lives of proteins with rapid turnover can be better appreciated by considering Millward's results (22) with muscle protein degradation. He found a tl2 of 6 d when radioactivity of aspartate and glutamate was followed (no reutilization) and a tl2 of 9.2 d when radioactivity of all other amino acids in muscle protein was followed (maximum reutilization). We believe, therefore, that the half-lives calculated here are reasonable approximations of the true values. It is also important to consider the related possibility that the food-induced changes in apparent degradation rate were artefactual, resulting from the changes in the degree of reutilization. Feeding might result in a decrease (by dilution) in the specific activity of labeled amino acids available for reutilization; thus, isotope reutilization might be less and apparent degradation faster. As shown in Fig. 6, however, the feeding period had no appreciable affect on the decline of amino acid specific activity. We believe that this observation and Circadian Rhythm of Sucrase Activity in Rats 1179 Downloaded from http://www.jci.org on June 18, 2017. https://doi.org/10.1172/JCI109772 our finding of circadian changes in degradation in fasted as well as fed animals (Fig. 5) exclude the possibility. Determination of protein synthetic rates in intestinal mucosa is difficult for a variety of reasons (21), and we have not attempted a study of sucrase-isomaltase synthesis at different times of the day. Our finding that incorporation of leucine into the protein (Table II) was not different between 0130 and 0930 h must be regarded as only supportive evidence against circadian changes in synthetic rates (although the data shown in Fig. 6 suggest that major changes in the leucine pool size may not occur during the time of the study). This evidence, however, is strengthened by consideration of the changes in enzyme activity. For example, from 0500 to 1100 h, one can calculate, from the specific activity of the pure enzyme and from the increase in enzyme activity, that total sucrase-isomaltase was increased by 0.77 or 0. 13 mg/h. Because degradation is essentially absent during these 6 h (t112 38 h), the value of 0.13 mg/h probably approximates the enzyme's true synthetic rate during that period. The value is quite similar to the synthetic rate of 0.11 mg/h found by us previously by analysis of enzyme mass and 24-h decay curves (7). Thus, the observed changes in sucrase-isomaltase protein appear to be compatible with the changes in degradation rates, and do not necessitate postulation of changing synthetic rates. Our concept of the relationship between the degradation rate and enzyme activity is shown in Fig. 7. From 0200 to 1100 h, degradation was almost nonexistent and enzyme levels increased to maximum; thereafter, degradation became feed l< > E24 cx a N. un 20 3 16 _ rapid and enzymne levels fell. Rapid degradation conltinued until 0200 h. Although Figs. 3-5 suggest that the degradationi of sucrase-isomiialtase is a biphasic process, it is possible that the rapid phase is characterized by more than one rate. This would explain our findling of a t112 of 6 h in the period from 1100 to 1700 h, and a t112 of 2.4 h in the periodl from 2000 to 0200 h. These calculationis were based Uponl analysis of two differenit experimiienits, however, and we believe that further studies with multiple time points within the rapid phase would be necessary to adequately evaluate this possibility. The obvious question is, What mechanismiis are responsible for the variations in degradation rates of suicrase-isomaltase? Sucrase-isomaltase is a relatively large protein (-210,000 mol wt) localized anatomnically and funictionally on the external surface of the brush border membranie (29, 30). Work by Alpers' laboratory (31, 32) suggests that this protein as well as other surface proteins may be in large part degraded intraluminally, with removal related to the presence of panicreatic enzymes. Thus, ligation of the pancreatic duct (31), pancreatectomy (32), and congenital acinar deficiency (33) all result in a decrease in degradation of large m11olecular weight brush border proteins. Intestinal mucosal stucrase activity is enhanced in these models as vell as in exocrine pancreatic insufficienicy in mnan (34). Finally, administration of cholecystokinin-pancreozymiiin is known to release sucrase into the intestinal lumeni (35). These in vivo studies are supported by in vitro work done by Alpers' laboratory, suggesting that pancreatic elastase will remove sucrase from the brush border membrane (31, 32). Thus, cyclical changes in these intraluminal factors could be responsible for alterations in the degradation rate of sucrase-isomaltase. It is interesting that in two other experimental models of altered sucrase activity, diabetes and sucrose feeding (7, 36-39), the mechanism also appears to be a change in the enzyme's degradation rate. Thus, the principal mleans of regulating sucrase-isomlaltase may be through changes in degradation. .> 12 H ACKNOWLEDGNIENTS U <,V 8 F U) ~3 ( 4 CL tl/2 38 h I fin E6 h 1 I | 0200 0500 0800 1100 1400 1700 2000 We are grateful to Dr. Robert WV. Swick for helpful discussioni and to Mr. Dan Omilianowski of the aimino acid analysis facility, University of Wisconsin Biophysics Laboratory, for performing the amino acid analyses. This study was supported by Nationial Institutes of Health grant AM-13927 and by Veterans Administration Research Funds. Time of Day (h) FIGURE 7 The relationship betwveen sucrase activity and half-life of the enzyme protein. Mean values for four animals ±SEM are given. Half-lives from the data presented in Fig. 3 are given. 1180 REFERENCES 1. Saito, M. 1972. Daily rhythmic changes in brush border enzymes ofthe small intestine and kidney in rat. Biochim. Biophys. Acta. 286: 212-215. 2. Saito, NI., E. MIurakami, and NI. Stida. 1976. Cireadian M. A. Kaufman, H. A. 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